Peptides and proteins commonly undergo a type of degradation reaction known as deamidation. This reaction occurs at susceptible glutaminyl and, especially, asparaginyl residues. Deamidation can occur at or near neutral pH by the beta-aspartyl shift mechanism, as well as at high or low pH, thereby making it difficult to prevent by simple buffer adjustment. Indeed, it is well known that the ubiquitous buffer component phosphate ion accelerates deamidation reactions, for example.
Protein/peptide deamidation is fundamentally different from a simple hydrolysis reaction. The first step in base-catalyzed deamidation of a peptide or protein at an asparaginyl or glutaminyl residue is usually nucleophilic attack of the adjacent main-chain nitrogen on the carbonyl, giving off an ammonia molecule, to form a short-lived intermediate which is a succinimide (a cyclic imide); this contrasts with the case of a simple hydrolytic attack of say, an ester or amide, by a water molecule (or one of its constituents, a hydroxyl ion or proton); in the latter case, the water molecule attacks an intact compound, whereas in peptide deamidation, water can attack the succinimide after the nucleophilic attack, so that attack by water is not the initial step. The facts that 1) the susceptible groups are the amides on the asparaginyl and glutaminyl residues; and 2) the attacking atom is a nitrogen, underscore an even more fundamental difference between peptide deamidation on the one hand, and de-esterification of ester-containing small molecules on the other. It is well established that in base-catalyzed deamidation, the formation of the succinimide by the afore-mentioned nucleophilic attack is the rate-limiting step. The activation energy for deamidation of proteins and peptides is approximately 22 kCal/mol. Those asparaginyl residues that are most prone to deamidation are those flanked on their C-terminal side by either a glycinyl or serinyl residue: Asn-Gly or Asn-Ser sequences.
Deamidation of an asparaginyl residue, yielding either an aspartate or isoaspartate residue (or, in uncompleted form, a succinimide derivative), is a change in the primary structure of the protein or peptide, and often results in significant loss of activity in vivo (viz., in a pharmaceutical preparation) and/or in vitro (e.g., in a diagnostic or assay system). Furthermore, such degradation of proteins and peptides can lead to an increase in immunogenicity, which can have disastrous effects. Also, deamidation can create degradation products that trigger abnormal changes in intracellular levels of certain peptides/proteins. And while the body has enzymes that repair deamidation damage in some proteins, the need for treatment with biopharmaceutical drugs in some cases relates right back to defective machinery in the functioning of these repair enzymes, so that administration with a (partially) deamidated protein or peptide could aggravate the exact problem the drug is supposed to treat. Deamidation of peptides and proteins can increase their incidence of denaturation or fibrillation, make them more prone to proteases, or modify their binding characteristics (e.g., of an antibody). As a particularly important example, succinimide intermediates from the first stages of base-catalyzed deamidation (particularly in the case of the peptide amylin) are hypothesized to be directly responsible for amyloid deposits, and thus may play central roles in such diseases as Parkinson's disease, type II diabetes, prion disease, and possibly Huntington's disease.
Degradation via deamidation is one of the reasons why most biopharmaceuticals must be produced in lyophilized (freeze-dried) form, requiring reconstitution before injection or other administration. Insulin is an extremely important example of a self-administered, home use drug that is supplied as a ready-to-use aqueous solution, and human insulin contains three asparagine residues. Deamidation of insulin is a well-established phenomenon. Recombinant human DNAase and recombinant soluble CD4 are well-established to lose activity upon deamidation.
Furthermore, even lyophilized formulations are sometimes formulated at very low pH (or less commonly, high pH) in order to limit deamidation and related reactions. This is in some cases due to deamidation that would otherwise occur during storage, or in other cases would occur between reconstitution and administration. Formulations at extreme values of the pH, namely less than about 4 or especially less than or equal to about 3, are highly unphysiological and can cause local damage, extravasation of the drug, and other harmful effects. Furthermore, restricting the range of pH available for formulating a particular peptide or protein can make it more difficult or even impossible to achieve targeted solubility or to avoid gelation, denaturation, clumping, etc. US patent application 2002/0061838 to Holmquist and Normady describes compositions with pH values between about 3.0 and 5.0 that contain acids, primarily in protonated form (i.e., formulation pH below the pKa of the acid, typically acetic acid), at small molar concentrations (typically 10 mM), intended to prevent aggregation or gelation of the peptide.
In aqueous solutions, the solution viscosity has only a relatively small effect on deamidation rates. However, the glassy and semicrystalline states that occur with lyophilization can strongly reduce deamidation, and thus the usual approaches of optimizing reducing sugars, non-reducing sugars, and polyols are very useful. Nevertheless, even in such formulations, deamidation—or at least the initial succinimide formation—can still occur within the lyo cake during storage as well as in pre-lyophilization production steps, and in the period between reconstitution and administration, which can be many hours in some cases, such as up to 8 hours in the case of the Alteplase formulation marketed as Activase®. The polyols, sugars and other cake-modulating compounds do not participate chemically to modify the deamidation reaction, but act indirectly through physical effects on the protein medium. Indeed, their stated main purpose is taken to be their effects on stabilizing secondary and tertiary structures of peptides and proteins, by reducing mobility of molecules or chemical moieities. For example, noted expert in the field John F. Carpenter, in the book Rational Design of Stable Protein Formulations: Theory and Practice (2002, Springer, John F. Carpenter and Mark C. Manning, eds.) states “Finally we will address this and other practical issues in the use of stabilizing excipients to inhibit protein unfolding during freezing and drying . . . . Among the numerous compounds tested, it appears that the most effective stabilizers of proteins during lyophilization are disaccharides.”
Human albumin solutions are used therapeutically as plasma volume expanders. Marketed albumin formulations for intravenous injection, such as Buminate® (Baxter Healthcare), Plasbumin® (Bayer Biological), and Human Albumin Grifols® (Grifols) contain equimolar amounts (1:1 molar ratio) of sodium caprylate and sodium N-acetyltryptophanate. The molar ratio of N-acetyltryptophan to albumin is 5.36:1, and since each albumin molecule has 17 asparaginyl residues, the ratio of N-acetyltryptophan to asparaginyl residues is 0.315:1. Shrake et al. [Vox Sang. 1984, 47(1), 7-18] have used differential scanning calorimetry to show that caprylate and acetyltryptophanate help prevent denaturation (disruption of protein secondary and/or tertiary structure) during the heat treatment step of albumin purification. Duggan and Luck [J. Biol. Chem. (1947) pp. 205-220] showed that these compounds—especially the caprylate—functioned by preventing viscosity rises in albumin solutions under conditions of denaturation with urea. According to that publication, “The comparative efficacy of stabilizers would then be determined by the mole ratio necessary to keep the viscosity of the albumin-urea system at its lowest value.”
It is well established [e.g., Bischoff and Kolbe (1994) J. Chromatogr. B, vol. 662, p. 261] that the deamidation-prone asparaginyl residues are those flanked on their C-terminal sides by either glycinyl or serinyl residues (small amino acids), and albumin has no such residue. Furthermore in its therapeutic role as a plasma volume expander, albumin's beneficial effect against hypovolemia would probably not be detrimentally affected by deamidation even if it were to occur.
OctreoScan® is a diagnostic preparation for the injection of a radiolabelled peptide derivative that contains, per 10 mL of reconstituted peptide-derivative solution, 2 mg of gentisic acid, 4.9 mg of trisodium citrate, 0.37 mg of citric acid, and 10 mg of inositol. The stated purpose of the gentisic acid in this formulation is to inhibit autoradiolysis of the radiolabelled compound capable of existing at a stable oxidation state. The peptide in OctreScan is derivatized first by covalent attachment of 4 acetic acid groups, and before administration by binding of indium-111. The amino acids in the peptide portion of the compound are phenylalanine, cystine, threonine, and tryptophan; thus, no asparaginyl or glutaminyl residues are present. The active moiety, namely the indium-111 atom, is chelated within the grasp of the acetic acid groups, analogously with the binding of multivalent ions with EDTA (ethylenediamine tetraacetic acid).
A list of proteins and peptides that undergo deamidation (non-enzymatically) has been compiled. [See T. Wright, Amino Acid Abundance and Sequence Data: Clues to the Biological Significance of Nonenzymatic Asparagine and Glutamine Deamidation in Proteins, in: Deamidation and Isoaspartate Formation in Peptides and Proteins, D. Aswad Ed., CRC Press, 1995; see also Teshima et al. in: Deamidation and Isoaspartate Formation in Peptides and Proteins, D. Aswad Ed., CRC Press, 1995]. This compilation will be taken as authoritative in this disclosure.
Human growth hormone is known to undergo deamidation degradation during the period (up to 14 days) between reconstitution and administration. Addition of zinc ions helps to ameliorate this and other degradation mechanisms of hGH and of insulin. Zinc in these formulations is, of course, a divalent cation.
Whereas a number of means are known in the art for protecting proteins in aqueous solution against physical changes such as aggregation, gelation, denaturation, molten states, thermal transitions, and generally, changes in secondary or tertiary structure, as well as for other types of chemical changes such as disulfide bond breakage or crosslinking (which can also lead to significant changes in physical structure and properties), the art has been lacking in broadly-effective and pharmaceutically-acceptable ways to specifically hinder or prevent non-enzymatic deamidation of asparaginyl residues. Methods that stabilize secondary and tertiary structures of proteins and peptides may play an indirect role in inhibiting deamidation reactions in isolated cases—though there is no a priori reason why the native conformation is the most stable against deamidation—and the complexes that can be promoted by multivalent ions can retard diffusivities and mobilities and, again, indirectly reduce deamidation rates, though often at the price of reducing drug solubility. More direct and widely-applicable stabilization of asparaginyl residues through chemical means are lacking.